It has been nearly three decades since the invention of DNA electrophoresis (1-5), and molecular biology laboratories still rely upon the separation of DNA, from plasmid DNA to PCR products, by use of denaturing or non-denaturing gel electrophoresis (1). Current conductive media for DNA electrophoresis are largely restricted to legacy Tris-acetic acid-disodium EDTA (TAE) and Tris-boric acid-disodium EDTA (TBE) at substantial ionic strengths, leading to higher cost and excessive heat generation and limiting the voltage and speed of electrophoretic runs (for description of buffers see Table 1). Investigators have compared and analyzed TAE and TBE buffers in DNA electrophoresis; however, to our knowledge no one has substantially investigated the simplification and substitution of components of these buffers to achieve a more efficient conductive medium for DNA electrophoresis (6). Conductive media for DNA electrophoresis derived essentially unchanged from RNA gel methodology, which in turn was adapted from a subset of buffers used for protein electrophoresis in the early 1960's (4, 7). The most common DNA electrophoretic media have contained Tris, an organic amine, as their primary cation; the first edition of Molecular Cloning (1982) contained a table of the three commonly used buffers for agarose gel electrophoresis: Tris-acetic acid-disodium EDTA, Tris-phosphate-disodium EDTA, and Tris-boric acid-disodium EDTA (1, 8). Presently, Tris-based buffers (TBBs) remain predominant in nearly all molecular biologic research and clinical laboratories (9). TBBs usually contain between 40 to 80 mM Tris (predominantly ionized), corresponding anion concentrations, as well as trace amounts of different forms of EDTA (1-2 mM), which could inhibit nucleases and certain enzymatic reactions. These high concentrations were historically supported by a preference to avoid ions of high mobility (7), to overcome detrimental effects on resolution of DNA-borate complexation by use of higher borate concentrations (6, 10), and to avoid the problems of dilute TAE media (10, 11). Tris is at times used with or replaced by other organic amines that buffer pH in the biological range.
It is well established that heat generation is a primary source of problems in gel electrophoresis, is responsible for sample diffusion, convection, denaturation, and poor gel integrity, and limits the ability to run gels at a high voltage (12). Ohm's law and the power law interrelate voltage (V), current (I), and power (P)(13, 14). Power consumed in the electrophoresis system manifests as heat; heat generation=P=VI. These interrelated variables are affected by ionic conductance due to choice of salts and ionized components in proportion to their particular concentrations in the media used in electrophoresis. The concentration of salts also determines the stability of the double-helical structure of DNA—a melted, single-stranded DNA (ssDNA) structure being desirable for certain DNA electrophoretic techniques.
There is a need in the art for improved conductive media for carrying out electrophoresis of nucleic acids.